Somatic embryo-derived plantlets of pines often fail to survive acclimatization, which limits commercial micropropagation. Conventional hardening methods do not correct the physiological weaknesses of in vitro plantlets, especially the lack of beneficial microbes. Here we developed a practical protocol for resistant Pinus elliottii. First, we used an optimized maturation protocol (three sequential ABA pre-treatments) and glucose for germination. Substrate screening showed that a peat:vermiculite:perlite mixture (3:1:1) gave the highest survival (98.9%). Then, before transplantation, we introduced a key bio-enhancement step: in vitro inoculation with the ectomycorrhizal fungus Pisolithus orientalis cfcc7668. This treatment achieved a mycorrhization rate of 97.7% and transformed root morphology from thin, sparsely branched roots to a coralloid, dichotomously branched system with a well-developed Hartig net. As a result, mycorrhizal plantlets had 100% transplant survival at 30 days and remained above 94% over 360 days, whereas non-inoculated controls dropped to 95.6% at 30 days and further declined to about 73% after three months. Pre-establishing ectomycorrhizal symbiosis effectively restores a key root function missing in in vitro plantlets. Our integrated procedure provides a practical method for clonal propagation of conifers.
Tian et al. (2026) studied this question.